A method for improving transgenic efficiency of cotton

A transgenic, cotton technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problems of offspring not conforming to Mendelian inheritance, long transformation period, transient expression and chimera, etc., to optimize stems. The effect of cutting-edge transformation technology system, shortening transformation cycle and reducing utilization value

Active Publication Date: 2016-08-24
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technical solution is mainly an in vitro shoot tip transformation method, without the process of cotton cell and tissue culture, the probability of chimerism increases, and the inheritance cannot be stable, and the offspring do not conform to the Mendelian inheritance law
However, many cotton transgenic methods (such as by hypocotyl transformation) through cell and tissue culture, because the hypocotyl will be induced and differentiated through callus, the transformation cycle is longer, generally 6-9 months ( Liu Mingyue, Optimization of Agrobacterium-mediated Cotton Genetic Transformation System, 2011, page 9), and is limited by the genotype of the transformed recipient
There are also gene gun transformation methods in which the shoot tip is the receptor, but compared with the Agrobacterium transformation method, the cost is high, and transient expression and chimerism occur
There are other Agrobacterium-mediated shoot tip transformation methods that show that it takes 3-4 months from co-cultivation to obtain resistant strains, which is a long period (Xu Dawei et al., 2011, China Agricultural Science Bulletin)

Method used

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  • A method for improving transgenic efficiency of cotton
  • A method for improving transgenic efficiency of cotton
  • A method for improving transgenic efficiency of cotton

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 improves cotton transgenic efficiency

[0029] 1 Materials and methods

[0030] 1.1 Test material

[0031] The test material used in this experiment is the upland cotton (G.hirsutum L.) variety: Jimian 14.

[0032] Transformation strain: Agrobacterium tumefaciens LBA4404.

[0033] The transformed gene GbAPX was derived from the root tissue transcriptome library of sea-island cotton variety Pima90-53, which was stressed by Verticillium dahliae, constructed in our laboratory.

[0034] 1.2 Main Reagents and Drugs

[0035] Acetosyringone, phytagel, plant growth regulators (IAA and KT), kanamycin and cephalosporin.

[0036] PCR primers were mainly synthesized by Sangon Bioengineering Co., Ltd.

[0037] 1.3 Test method

[0038] 1.3.1 Culture medium preparation

[0039] Prepare the required media according to Table 1.

[0040] The medium used in the test in table 1

[0041]

[0042]

[0043] 1.3.2 Aseptic vaccine culture

[0044] Peel the cottonsee...

Embodiment 2

[0068] Example 2 Co-cultivation of transgenic cotton and optimization of screening medium

[0069] The operation method is the same as in Example 1, except that the difference from Example 1 is that in this example, the shoot tips of sterile cotton seedlings are infected with Agrobacterium and placed on co-cultivation medium and screening medium of different hormones (combinations) for growth. Specifically:

[0070] The shoot tips of sterile cotton seedlings were infected by Agrobacterium and placed on the co-cultivation medium and screening medium of different hormones (combinations), 2 bottles of each hormone (hormone combination), 46 bottles in total (including 2 bottles of control) , 5 stem tips per bottle.

[0071] The co-cultivation medium is: MSB 5 +hormone (combination)+200μmol AS+30g / L glucose+2.4g / L phytagel, pH5.4;

[0072] The screening medium is: MSB 5 +200mg / Lcef+80mg / L kan+glucose 15g / L+sucrose 15g / L+hormone (combination)+0.25% Phytagel, pH5.8;

[0073] The...

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Abstract

The invention provides a method for improving the transgenic efficiency of cotton. The method comprises the following steps: a. Infecting the shoot tip of the aseptic cotton seedling with the plant expression vector containing the target gene by using the Agrobacterium-mediated method, and the infected Cotton shoot tips are placed on the co-cultivation medium for co-cultivation; b. After co-cultivation, the cotton shoot tips are transferred to the screening medium for cultivation; both the co-cultivation medium and the screening medium contain a concentration of 0.3mg / L of KT and a concentration of 0.2mg / L of IAA. Utilize the method of the present invention to carry out aseptic seedling culture time 2-3 days, co-culture 2 days, screening culture 15-20 days, rooting culture 20 days, transformation cycle is no more than 2 months, and positive transformation rate can reach 8 after screening. %.

Description

technical field [0001] The invention relates to the technical field of cotton transgenes, in particular to a method for improving the efficiency of cotton transgenes. Background technique [0002] Significant progress has been made in the genetic improvement of cotton using genetic engineering technology. In 1987, Umbeck et al first reported the transfer of kanamycin-resistant gene into cotton by Agrobacterium-mediated method (Umbeck P. Genetically transformed cotton (Gosspium hirsutum L.) Plants. Bio / Teehnology, 1987, 5: 263-266). The traditional genetic transformation method has a long transformation period, strong genotype dependence of plant regeneration, high deformity rate of regenerated seedlings, and low survival rate of transplanting, which has become a bottleneck restricting the research of cotton transgenic breeding. [0003] Cotton shoot tips have the characteristics of direct differentiation and regeneration, once transformed, the formation of leaves and roots ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 吴金华王省芬张桂寅吴立强李志坤张艳王国宁柯会锋马峙英
Owner HEBEI AGRICULTURAL UNIV.
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